The brain derived neurotrophic factor (BDNF) gene hastwo polyadenylation sites, leading to the generation of two mRNAs, one with a short 3'untranslated region (UTR) and another with a long 3'UTR. Previous results have demonstrated that the long BDNF 3'UTR is sufficient to target mRNAs to dendrites. It has also been demonstrated that BDNF is required for long-term potentiation (LTP) that is dependent on protein synthesis. We hypothesize that dendritically localized BDNF mRNAs are basally repressed until local synaptic activity signals an increase in BDNF translation, allowing for activity-dependent modulation of the synapse. In this study we propose to investigate the mechanisms by which this basal repression is regulated. We will first establish whether local BDNF translation is increased in response to activity by looking at changes in endogenous BDNF levels in synaptoneurosomes after stimulation with KCI or glutamate using radioimmunoprecipitation to detect new protein synthesis, as well as by optically monitoring the expression of a fluorescent reporter linked to either the short or long BDNF 3'UTR transfected into primary hippocampal cultures under conditions of chemical LTP (cLTP). In order to further elucidate the mechanisms mediating activity-dependent increases in local translation we will repeat the cLTP experiments with the co-application of various inhibitors of pathways known to be important for the establishment of LTP. We will then use primary hippocampal cultures to probe the importance of a brain-expressed miRNA that has multiple putative binding sites in the long BDNF 3'UTR in suppressing the translation of dendritic mRNAs containing the BDNF 3'UTR. This will be done in cultures transfected with the BDNF 3'UTR reporters by overexpressing the miRNA and assaying for a decrease in reporter signal, and by knocking down the activity of the miRNA through the use of antisense oligonucleotides and mutation of the miRNA binding sites on the reporter and looking for an increase in reporter signal. In order to link synaptic activation and miRNA de-repression we will repeat the experiments overexpressing and knocking down miRNA activity in the presence of cLTP stimulation. Since BDNF is important in adult synaptic plasticity, and has been shown to be therapeutic in promoting neuronal survival in models of neurodegeneration, understanding the mechanisms that regulate its expression in dendrites could allow for discovery of potential targets for drug therapies to treat psychiatric disorders, such as major depression, as well as neurodegenerative disorders, such as Alzheimer's, Parkinson's and Huntington's disease.
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